CN115112776B - Combined marker, application thereof in diagnosing atrial fibrillation and diagnostic reagent or kit - Google Patents
Combined marker, application thereof in diagnosing atrial fibrillation and diagnostic reagent or kit Download PDFInfo
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Abstract
The invention relates to a novel application of lipid metabolites of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 in preparing a kit for diagnosing atrial fibrillation diseases as combined markers in a plasma sample. The invention also relates to a kit for detecting atrial fibrillation in a subject suffering from arrhythmia, by detecting the respective concentrations of the above-mentioned combined markers in a plasma sample from the subject, calculating the combined marker variable Prob based on a binary logistic regression equation and determining the cut-off value (cut-off value), and determining whether the subject suffers from atrial fibrillation. The kit can realize high-sensitivity and high-efficiency detection of atrial fibrillation diseases. The three lipid metabolites related by the invention have the characteristics of low detection cost and good stability. The combination of the above lipid metabolites can be used in the auxiliary diagnosis of atrial fibrillation.
Description
Technical Field
The present invention relates to the field of analytical chemistry and clinical medicine. Specifically, the invention relates to a kit for distinguishing atrial fibrillation by using Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 as joint markers.
Background
Atrial fibrillation is the main causative agent of cardiovascular disease, greatly increasing the medical costs and mortality of cardiovascular disease. In recent years, the incidence of atrial fibrillation and mortality have increased, particularly in the elderly. Atrial fibrillation is one of the most common supraventricular arrhythmias characterized by uncoordinated electrical activation of the atria and an irregular, rapid ventricular response leading to hemodynamic damage. Atrial fibrillation is not adequately diagnosed or treated and can have a promoting effect on the development of cardiomyopathy, heart failure and stroke (document 1:Cecilia,Gutierrez,Daniel G,et al.Diagnosis and Treatment of Atrial Fibrillation [ J ]. American family physician,2016,94 (6): 442-452.). In view of the development of portable intelligent devices, the implantation of specialized algorithms therein may also enable diagnosis and detection of atrial fibrillation, but such techniques are costly and require noiseless tracking for optimal performance. This can be difficult when the device is provided for use by a patient or community. Although the pulse rate is sensitive to diagnosis of atrial fibrillation, it is not specific and ultimately requires an electrocardiogram to confirm suspected atrial fibrillation. Currently 12-lead and single-lead electrocardiographic recordings of p-waves are used for the definitive diagnosis of atrial fibrillation, but such long-term continuous heart rate monitoring by external devices is limited in cost (documents 2:Freedman B,Camm J,Calkins H,et al.Screening for Atrial Fibrillation AReport of the AF-SCREEN International Collaboration [ J ]. Circulation,2017,135 (19): 1851-1861.). Thus, the identification of more reliable plasma biomarkers is of great importance for the clinical diagnosis of atrial fibrillation.
Metabonomics is a specialized discipline that describes changes or time-dependent changes in metabolites of an organism by examining the organism after it has been stimulated or disturbed. Studies have shown that the occurrence and development of atrial fibrillation is closely related to a variety of small molecule metabolite disorders, such as purine metabolism, lipid metabolism, d-glutamine and d-glutamate metabolism, and the like. The invention detects lipid metabolites in blood plasma by using a metabonomics method of liquid chromatography-mass spectrometry, screens target metabolites through bioinformatics analysis, and is expected to be applied to diagnosis of atrial fibrillation diseases. In addition, the chromatography-mass spectrometry technology provides a rapid, sensitive, stable and low-cost detection method for detecting the small molecule metabolites. Xuan Qiu Hui et al define a combination marker of 12-hydroxyeicosatetraenoic acid (12-HETE) and 2-piperidone serum based on a multi-platform metabonomics study, which can discriminate between Diabetic Retinopathy (DR) and diabetes mellitus very well, especially with high sensitivity in early DR detection (literature 3:Xuan Q,Ouyang,Y,et al.Multiplatform Metabolomics Reveals Novel Serum Metabolite Biomarkers in Diabetic Retinopathy Subjects[J ]. Advanced Science,2020,7 (22): 2001714.). Various analytical techniques are currently used for metabonomic detection, such as nuclear magnetic resonance, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, capillary electrophoresis chromatography-mass spectrometry, etc., wherein the application of liquid chromatography-mass spectrometry is increasingly mature and widespread.
The invention utilizes ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) technology to detect and analyze the blood plasma of a batch of ventricular premature beat patients, supraventricular tachycardia patients and auricular fibrillation patients, and divides the samples into auricular fibrillation groups and non-auricular fibrillation groups (including the ventricular premature beat patients and the supraventricular tachycardia patients). Multiple preference, a panel of combined markers of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5, and Phosphatidylinositol (PI) 16:0_18:1 was determined for use in one-time diagnosis of atrial fibrillation patients in arrhythmic subjects. Fatty Acids (FA) are involved in the energy metabolism of cardiomyocytes as an important class of energy substrates (documents 4:Harada M,Melka J,Sobue Y.Metabolic Considerations in Atrial Fibrillation-Mechanistic Insights and Therapeutic Opportunities [ J ]. Circulation Journal,2017,81 (12): 1749-1757.). Lysophosphatidylcholine (LPC) plays a protective role in the anti-inflammatory response and in the progression of metabolic diseases in the body (documents 5:Taylor L A,Arends J,Hodina A K,et al.Plasma lyso-phosphatidylcholine concentration is decreased in cancer patients with weight loss and activated inflammatory status [ J ]. Lipids in Health and Disease,2007,6 (1): 17-34.). Phosphatidylinositol (PI) is an important class of compounds involved in cell signaling and arachidonic acid synthesis, and conversion of arachidonic acid to prostaglandins and thromboxanes by cyclooxygenase accelerates the development of inflammation (document 6:Samuelsson B.Leukotrienes:mediators of immediate hypersensitivity reactions and inflammation[J ]. Science,1983,220 (4597):568-575.). There is no report on the use of the above combined markers for diagnosis of atrial fibrillation.
Disclosure of Invention
The invention aims at solving the problem that the diagnosis of atrial fibrillation of arrhythmia population is difficult, and provides a novel plasma lipid combined marker which can be applied to the diagnosis of atrial fibrillation of arrhythmia patients and an analysis and detection method for the combined marker.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
(1) Performing metabonomics fingerprint analysis on the plasma of patients suffering from ventricular premature beat, supraventricular tachycardia and atrial fibrillation by using a metabonomics technology of high performance liquid chromatography-mass spectrometry;
(2) Non-parametric testing of quantifiable metabolites using MEV software, calculating false positive rate (FDR) values and p values for all metabolites, with significant differences for metabolites with FDR values <0.2 and p values <0.05, thus finding significant differences for 45 metabolites in non-atrial fibrillation (ventricular premature and supraventricular tachycardia) and atrial fibrillation patients;
(3) Using data statistics software SPSS, by binary logistic regression analysis method, by forward: conditional methods 45 differential metabolites were screened, regressed to a combined marker variable, and then the sensitivity and specificity of the combined markers were assessed using a ROC (receiver operating characteristic) curve. The sensitivity and the specificity are both higher, meanwhile, the combination with simplicity (namely, the number of metabolites involved in the combination is smaller) can be used as a combined marker, and Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 can be used as combined markers for assisting in diagnosing atrial fibrillation diseases;
(4) Validating the combined markers using another set of plasma samples of ventricular premature, supraventricular tachycardia and atrial fibrillation patients to determine that Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 can be used as combined markers to aid in diagnosing atrial fibrillation diseases;
(5) Use of a combination marker: fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5, and Phosphatidylinositol (PI) 16:0_18:1 concentrations were reduced in the plasma of patients with atrial fibrillation relative to patients with non-atrial fibrillation (ventricular premature and supraventricular tachycardia). The metabolites are regressed into a joint marker variable p by a binary logistic regression method by using data statistical software SPSS, and a binary logistic regression equation is as follows:
p＝1/[1+e -(-161.307*a-2.319*b-18.907*c+6.716) ]
wherein a is the concentration of Fatty Acid (FA) 23:0 in a plasma sample, b is the concentration of Lysophosphatidylcholine (LPC) 20:5 in a plasma sample, c is the concentration of Phosphatidylinositol (PI) 16:0_18:1 in a plasma sample. The resulting variable p is elevated in patients with atrial fibrillation, and the value of this variable can be used to assist in determining atrial fibrillation. The intercept value of the combined marker for atrial fibrillation judgment determined by the invention is set to 0.453, and if the intercept value is higher than the intercept value, atrial fibrillation is possible.
(6) The diagnostic system comprises: the chromatographic column is a Waters BEH C8 column (100 mm×2.1mm,1.7 μm) (Waters, milford, mass.), the separation system is Agilent 1290 information II LC, the detection system is Agilent 6546Q-TOF mass spectrum, and positive ion mode and negative ion mode detection are used;
(7) Determining the optimal composition of the kit:
a. standard chemicals: fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1. The standards were used for characterization of small molecule metabolites Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1, respectively, in plasma. Carrying out liquid chromatography-mass spectrometry on three substance standard substances with the concentration of 5-20 mug/mL, determining the chromatographic retention time of the three standard substances and the actually measured mass-to-charge ratio of three ions, and comparing the chromatographic retention time and the actually measured mass-to-charge ratio with the three substances actually measured in a sample of a subject;
b. extract for pretreatment of plasma samples: the extract was used to pre-treat plasma samples from subjects as two internal standard isopropanol solutions containing 0.56 μg/mL D3-Fatty Acid (FA) 18:0 and 0.28 μg/mL Lysophosphatidylcholine (LPC) 19:0. Internal standard D3-Fatty Acid (FA) 18:0 is used to correct Fatty Acid (FA) 23:0 and Phosphatidylinositol (PI) 16:0_18:1, lysophosphatidylcholine (LPC) 19:0 is used to correct Lysophosphatidylcholine (LPC) 20:5. Comparing the ionic peak intensities of the three substances after qualitative treatment in each subject sample with internal standard substances in the extracting solution, and obtaining relative concentrations of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 through internal standard correction;
c. eluent: mobile phase a was a 60% (v/v) acetonitrile in water containing 10mM ammonium acetate; mobile phase B was a 90% (v/v) isopropanol acetonitrile solution containing 10mM ammonium acetate;
the invention also relates to a kit for detecting atrial fibrillation in a subject suffering from arrhythmia, by detecting the respective concentrations of the above-mentioned combined markers in a plasma sample from the subject, calculating the combined marker variable Prob based on a binary logistic regression equation and determining the cut-off value (cut-off value), and determining whether the subject suffers from atrial fibrillation. The kit can realize high-sensitivity and high-efficiency detection of atrial fibrillation diseases. The three lipid metabolites related by the invention have the characteristics of low detection cost and good stability. The combination of the above lipid metabolites can be used in the auxiliary diagnosis of atrial fibrillation.
The invention has the following effects:
the combined marker variable p in the plasma can have good diagnosis on atrial fibrillation. The detection kit provided by the invention has the advantages of simplicity, convenience, rapidness and good repeatability in detecting the lipid metabolite combination, and is suitable for assisting the clinical diagnosis of atrial fibrillation. Sensitivity and specificity and area under the curve (AUC) are shown in table 1 below.
TABLE 1 results of use of the combination markers
Drawings
FIG. 1. Relative content changes (mean.+ -. Standard deviation) of Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5, and Phosphatidylinositol (PI) 16:0_18:1 in non-atrial fibrillation patients (ventricular premature and supraventricular tachycardia) and in atrial fibrillation patients in the discovery and validation set.
Fig. 2. (a) ROC plots, auc= 0.831, for combination markers in the discovery set for diagnosing atrial fibrillation; (B) The combined markers were used in the validation set for ROC profile diagnosis of atrial fibrillation, auc=0.745.
Detailed Description
Example 1
1. Plasma sample collection
All volunteers enrolled in the study signed informed consent prior to plasma sample collection. Blood samples of 49 patients suffering from atrial fibrillation, 23 patients suffering from ventricular premature beat and 23 patients suffering from supraventricular tachycardia were collected under the same conditions in an anticoagulant tube, centrifuged at 4500rpm/min for 5 minutes after collection, and then the blood plasma was taken and stored in a-80 ℃ refrigerator, respectively, for later use.
2. Analysis method
2.1 plasma sample pretreatment
First a 96-well protein precipitation plate was placed over a 96-well receiving plate. To a 96-well protein precipitation plate was added 360. Mu.L of isopropyl alcohol containing an internal standard (containing 2 internal standards: 0.56. Mu.g/mL D3-Fatty Acid (FA) 18:0 and 0.28. Mu.g/mL Lysophosphatidylcholine (LPC) 19:0), followed by 40. Mu.L of plasma. The protein precipitation plate and receiving plate were shaken for 10 minutes and then centrifuged at 500g for 10 minutes. The filtrate is centrifuged in a receiving plate below and separated from the proteins in a settling plate above. The upper plate containing the precipitate was discarded, and 40. Mu.L of the sample was taken in the lower plate, diluted with 80. Mu.L of acetonitrile/isopropyl alcohol/water solution in a volume ratio of 65/30/5, and after shaking for 5 minutes, positive ion mode detection was performed. The remaining samples were directly subjected to negative ion mode detection.
2.2 instrument conditions
The liquid chromatography system used Agilent 1290 information II LC (Agilent Technologies Inc, california, USA). Chromatographic column: waters BEH C8 column (100 mm×2.1mm,1.7 μm) (Waters, milford, MA), column temperature: 60 ℃, flow rate: 0.3ml/min. Mobile phase: 60% (v/v) acetonitrile in water of 10mM ammonium acetate (phase A) and 90% (v/v) isopropanol in acetonitrile of 10mM ammonium acetate (phase B). Gradient: the initial gradient was 50% B for 1.5min, followed by a linear increase to 85% B over 7.5min, then mobile phase B increased to 100% over 0.1min and for 1.9min. Then reduced to 50% b in 0.1min and maintained for 1.9min.
The detection systems were Agilent 6546Q-TOF mass spectrometry (Agilent Technologies Inc, california, USA), positive ion mode and negative ion mode. TOF full scan range m/z 100-1200; gas temperature 320 ℃, drying gas flow rate 8L/min, atomizing gas 39psig, sheath gas temperature 350 ℃, sheath gas flow rate 11L/min, positive ion mode voltage 4000V, negative ion mode voltage 3000V, mixed collision energy 15eV and 30eV, MS 2 The scanning range m/z is 100-1200, and the secondary data acquisition adopts an iterative acquisition mode.
3. Plasma test result and auxiliary diagnosis method
The peak areas of the combined markers Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 and the internal standard compound are respectively extracted, the peak areas of the metabolites are subjected to internal standard correction to obtain corresponding relative intensities, the Fatty Acid (FA) 23:0 and the Phosphatidylinositol (PI) 16:0_18:1 are corrected by adopting D3-Fatty Acid (FA) 18:0, and the Lysophosphatidylcholine (LPC) 20:5 is corrected by adopting Lysophosphatidylcholine (LPC) 19:0. Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 were quantitatively analyzed. The relative amounts of the above metabolites (i.e., the above relative intensities) in the non-atrial fibrillation group (ventricular premature and supraventricular tachycardia) and the atrial fibrillation group are shown in fig. 1 (discovery set) and table 2.
TABLE 2 relative amounts of Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 in the non-atrial fibrillation group (ventricular premature and supraventricular tachycardia patients) and in atrial fibrillation
Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 levels were down-regulated in the plasma of patients with atrial fibrillation relative to non-atrial fibrillation patients (ventricular premature and supraventricular tachycardia). Meanwhile, the relative content of each metabolite is substituted into SPSS software to carry out binary logic modeling analysis, and a regression equation of the built model is as follows:
p＝1/[1+e -(-161.307*a-2.319*b-18.907*c+6.716) ]
the regression equation builds a model based on e, where e is a natural constant (Euler number), a is the concentration of Fatty Acid (FA) 23:0 in the plasma sample, b is the concentration of Lysophosphatidylcholine (LPC) 20:5 in the plasma sample, and c is the concentration of Phosphatidylinositol (PI) 16:0_18:1 in the plasma sample. The resulting variable p is elevated in patients with atrial fibrillation, and the value of this variable can be used to assist in determining atrial fibrillation. The intercept value of the combined marker for atrial fibrillation judgment determined by the invention is set to 0.453, and if the intercept value is higher than the intercept value, atrial fibrillation is possible. For non-atrial fibrillation diseases (ventricular premature beat patients and supraventricular tachycardia patients) and atrial fibrillation diseases, the small-molecule lipid combination marker has better discrimination capability and better diagnosis effect. Auc= 0.831, sensitivity was 83.7% and specificity was 71.7% (see table 2 and fig. 2).
Table 3.
Example 2
1. Plasma sample collection
All volunteers enrolled in the study signed informed consent prior to plasma sample collection. Blood samples of 36 patients with atrial fibrillation, 10 patients with ventricular premature beat and 18 patients with supraventricular tachycardia were collected under the same conditions in an anticoagulant tube, centrifuged at 4500rpm/min for 5min after collection, and then the plasma was taken and stored in a refrigerator at-80 ℃ for later use.
2. Analysis method
Same as in example 1
3. Plasma test result and auxiliary diagnosis method
The relative amounts of Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 in the non-atrial fibrillation group (ventricular premature and supraventricular tachycardia) and in the atrial fibrillation group are shown in FIG. 1 (validation set) and Table 4.
TABLE 4 relative amounts of Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 in non-atrial fibrillation groups (ventricular premature and supraventricular tachycardia patients) and atrial fibrillation
The relative concentrations of the metabolites were substituted into the binary logistic regression equation obtained in example 1 and diagnostic effect discrimination was performed using the cut-off values obtained in example 1. For non-atrial fibrillation diseases (ventricular premature beat and supraventricular tachycardia) and atrial fibrillation diseases, the small-molecular lipid composite marker has better discrimination capability and better diagnosis effect. Auc=0.745, sensitivity 77.8% and specificity 67.9% (see table 3 and fig. 2).
Table 5.
The second set of validation results substantially match the first set of findings. Fatty Acids (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 are useful as combined markers for diagnosing atrial fibrillation disease in a subject, with good sensitivity and specificity. The kit has the characteristics of low detection cost and good stability, and has good development and application values.
It is to be understood that while the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
Claims (6)
1. A combination marker for diagnosing an atrial fibrillation patient in an arrhythmic subject, the combination marker consisting of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5, and Phosphatidylinositol (PI) 16:0_18:1.
2. A diagnostic kit for diagnosing patients with atrial fibrillation in a subject suffering from arrhythmia,
(1) Standard chemicals: fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1, wherein the standard chemicals are respectively used for characterization of corresponding small-molecular lipid metabolites of blood plasma, namely Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1, and the concentration is 5-20 mug/mL;
(2) Extract containing internal standard compound: the extract is used for preprocessing a plasma sample from a subject and is an isopropanol solution containing 0.42-0.7 mug/mLD 3-Fatty Acid (FA) 18:0 and 0.16-0.4 mug/mL Lysophosphatidylcholine (LPC) 19:0.
3. The diagnostic kit of claim 2, further comprising an eluent that is: the mobile phase A is a 55% -65% acetonitrile aqueous solution containing 8.5-11.5 mM ammonium acetate; the mobile phase B is an 85% -95% isopropanol acetonitrile solution containing 8.5-11.5 mM ammonium acetate.
4. Use of a combination marker according to claim 1 consisting of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 in the preparation of a diagnostic reagent or kit for diagnosing an atrial fibrillation patient in a subject suffering from an arrhythmia.
5. The use according to claim 4, wherein the diagnostic reagent or kit is a combination of reagents comprising the detection of the relative concentrations of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1 in the plasma of the subject using a liquid chromatography-mass spectrometer.
6. The use of claim 5, wherein the means for detecting the relative concentrations of Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5, and Phosphatidylinositol (PI) 16:0_18:1 in the plasma of the subject using a liquid chromatograph-mass spectrometer comprises:
(1) Standard chemicals: fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1, wherein the standard chemicals are respectively used for characterization of corresponding small-molecular lipid metabolites of blood plasma, namely Fatty Acid (FA) 23:0, lysophosphatidylcholine (LPC) 20:5 and Phosphatidylinositol (PI) 16:0_18:1, and the concentration is 5-20 mug/mL;
(2) Extract containing internal standard compound: the extracting solution is used for preprocessing a plasma sample from a subject, and is an isopropanol solution containing two internal standards of 0.42-0.7 mug/mL D3-Fatty Acid (FA) 18:0 and 0.16-0.4 mug/mL Lysophosphatidylcholine (LPC) 19:0;
(3) Eluent: mobile phase a was a 60% v/v acetonitrile in water containing 10mM ammonium acetate; mobile phase B was a 90% v/v isopropanol acetonitrile solution containing 10mM ammonium acetate.
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